Johne's disease is a chronic gastroenteritis affecting ruminant animals worldwide with national prevalence rates of 32% in Canada, and 68% in the U.S. (1, 2). Calves are most susceptible and once colonized they remain asymptomatic for 2 to 5 years while shedding M. avium subsp. paratuberculosis (MAP) in feces, colostrum, and milk (3). As there is no effective or approved treatment for Johne's disease, control of MAP at the herd-level requires identification of infected animals, specifically MAP-shedders, and their removal from the herd (4). In addition, certain calf rearing, cleaning, and animal husbandry practices have shown promise of reducing MAP prevalence (5). To accurately detect MAP-infected animals a number of methodologies have been employed including serodiagnosis by ELISA, detection by PCR amplification of MAP-specific sequences in feces and milk, measurement of interferon-gamma secretion by T-cells, and bacterial culture of fecal and milk samples. However, variables such as antigen-specificity and sensitivity, reproducibility, time to colony detection, PCR inhibitors in feces and milk, and cross-reactivity are all factors that currently limit the efficacy of these methods (6). Serodiagnosis by ELISA has been recommended as the primary methodology as this platform is relatively less laborious, rapid, cost-effective, and the results are simple to interpret (7).
Commercially available ELISAs for Johne's disease have high specificity (90-99%), but all suffer from low sensitivity (13.5% to 42%) (8). The antigen compositions of these ELISAs are mostly proprietary in nature, but have been shown to contain a mixture of cellular proteins and/or purified protein derivative (9). The sensitivity of these assays was reported to be lowest (5-7%) in cattle shedding low levels of MAP, and only reached sensitivity values of 70-80% when high levels of MAP are detected in feces (10). Moreover, preabsorption of serum with Mycobacterium phlei crude protein lysates has improved the specificity of commercial ELISAs by removing cross-reactive antibodies (11).
The sensitivity of serodiagnostics improved with the use of MAP culture filtrate (CF) proteins, and similarly for other mycobacterial pathogens including M. bovis and M. tuberculosis (6, 12, 13). Compared to cellular proteins, MAP CF proteins showed greater reactivity with serum from MAP-infected cattle both with respect to the number of antigens detected and in the intensity of reaction on immunoblots (14). The use of MAP CF antigens in ELISAs increased assay sensitivity by 25% over commercial ELISAs in low MAP-shedding cattle (15). However, antigen selection remains a challenge, as there is no single MAP-specific antigen that is recognized by all infected cattle, especially those in early and subclinical stages of disease. Recently, early serodiagnosis was addressed using experimentally infected calves, and screening for antibody responses to a panel of 96 recombinant MAP-antigens (16). Antibody responses were detected as early as 70 days post-infection; however, fluctuations in antibody response and epitope specificity were observed over 321 days (16). These data suggest the need for a standardized cocktail of antigens for incorporation into a single ELISA for detection at all stages of disease in infected cattle.
There remains a need for new and improved biomarkers for MAP as well as new and improved methods for identifying animals with MAP infections and/or Johne's disease.